- Volume 71, Issue 10, 1990
Volume 71, Issue 10, 1990
- Animal
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Production of a non-functional nef protein in human immunodeficiency virus type 1-infected CEM cells
The nef gene product of the human immunodeficiency virus (HIV) is suggested to be a negative factor involved in down-regulating viral expression by a mechanism in which the correct conformation of the nef protein is essential. The nef protein expressed by vaccinia virus recombinants is phosphorylated by protein kinase C. We investigated the synthesis of the nef protein and its state of phosphorylation during HIV-1 infection of a T4 cell line (CEM cells). Maximum synthesis of viral proteins occurred 3 days after infection, when more than 90% of cells were producing viral proteins. The synthesis of the nefprotein was detected in parallel with the env and gag proteins. As expected, the nef protein was myristylated but not phosphorylated, and its half-life was less than 1 h. By the use of the polymerase chain reaction technique, we isolated and sequenced the nef gene of this HIV-1 stock. Two significant mutations were observed. Firstly, threonine, at amino acid number 15, the site of phosphorylation by protein kinase C, was mutated into an alanine, and secondly aspartic acid of the tetrapeptide WRFD, which is probably involved in GTP binding, was mutated into an asparagine. The mutated nef gene was expressed in a vaccinia virus system, in which it was not phosphorylated and its half-life was dramatically reduced compared to the wild-type nef gene product. Furthermore, down- regulation of CD4 cell surface expression was no longer affected by the mutated nef gene. These results emphasize that phosphorylation of the nef protein provides an efficient test to monitor its biological activity.
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Inhibition of the uncoating of bovine enterovirus by short chain fatty acids
More LessShort chain fatty acids inhibit the replication of bovine enterovirus but are almost ineffective against poliovirus type 1, coxsackievirus B5, encephalomyocarditis virus and human rhinovirus 1B. Lauric acid binds to bovine enterovirus, thereby stabilizing the virus particle to heat degradation. Fatty acid-bound virions attach to susceptible cells but fail to undergo cell-mediated uncoating. The inhibitory effect is reversible with chloroform and may result from a hydrophobic interaction between the fatty acid and a specific site on the virus particle.
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The complete nucleotide sequence of enterovirus type 70: relationships with other members of the Picornaviridae
Enterovirus type 70 (EV70) is the causative agent of acute haemorrhagic conjunctivitis and may also give rise to a rare neurological complication closely resembling poliomyelitis. The complete nucleotide sequence of the genome of EV70 has been determined from cDNA cloned in Escherichia coli. The genome consists of a 5′ non-coding region of 726 nucleotides (nt), a long open reading frame of 6582 nt and a 3′ non-coding region of 82 nt prior to the poly(A) tract. Comparison of the nucleotide sequence and the predicted amino acid sequence of the polyprotein with those published for other enteroviruses reveals sufficiently high similarity to predict antigenic regions and polyprotein cleavage sites. The P1 region of EV70 is as similar to those of the entero- as to those of the rhinoviruses, whereas the P2 and P3 regions are more closely related to the coxsackie B and swine vesicular disease viruses than other entero- or rhinoviruses.
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Attenuation of wild-type yellow fever virus by passage in HeLa cells
During the 1960s three different research groups reported that passage of wild-type yellow fever (YF) virus [strain Asibi (YF-Asibi)] in HeLa cells resulted in attenuation of the virus for monkeys so that the virus no longer caused viscerotropic disease. We have repeated and extended this observation to analyse the process of attenuation of YF virus during cell culture passage. A large plaque (LP) variant of YF-Asibi virus became attenuated for both monkeys and mice following six serial subcultures in HeLa cells (YF-Asibi-LP HeLa p6). Thus, attenuation was probably due to a genetic change in the virus population rather than to selective enrichment of a pre-existing variant of YF-Asibi-LP virus. No evidence was obtained to implicate defective interfering particles in the attenuation process. Comparison of the YF-Asibi-LP viruses before and after passage in HeLa cells, using a panel of envelope protein-reactive monoclonal antibodies (MAbs), showed that MAbs which specifically neutralize YF-Asibi-LP virus, and not YF 17D-204 vaccine virus, also neutralized YF-Asibi-LP HeLa p6. This indicated that the epitopes involved in the biological process of neutralization were not altered during attenuation. However, two MAbs that recognize envelope protein epitopes did distinguish between HeLa- and non-HeLa-passaged YF-Asibi-LP virus. One of these (MAb 117) which is YF wild-type-specific, recognized YF-Asibi-LP virus but not YF-Asibi-LP HeLa p6 virus, whereas the other (MAb 411), which is YF vaccine-specific, recognized YF-Asibi-LP HeLa p6 virus but not YF-Asibi-LP virus. These results suggest that antigenic changes in the viral envelope protein may determine the relative virulence or attenuation of YF virus.
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Generation and transmission of Rift Valley fever viral reassortants by the mosquito Culex pipiens
More LessReassortant viruses containing heterologous S and M genomic RNA segments were obtained from both mosquito and vertebrate hosts that had been co-infected with Egyptian and Senegalese strains of Rift Valley fever (RVF) virus. The origin of the S and M RNA segments in each plaque-cloned virus was determined with monoclonal antibodies capable of differentiating the nucleocapsid protein (S segment marker) or the G1 glycoprotein (M segment marker) of the parental strains. In the mosquito Culex pipiens, reassortants were detected after sequential ingestion of parental viruses by interrupted feeding on two infected hamster hosts, after feeding on a single host that had been infected with both parental strains, and from individual mosquitoes inoculated intrathoracically with both parental strains. Reassortant viruses replicated efficiently in mosquitoes and were readily transmissible by bite to hamsters. Replication of a second infecting strain of RVF virus was, however, completely inhibited if that virus was inoculated into a mosquito ⩾48 h after the first viral strain. Genetic reassortment may provide a mechanism for increased heterogeneity, and thus affect the epidemiology and evolution of RVF virus.
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Morphological studies of the neutralization of influenza virus by IgM
More LessQuantitative relationships between neutralization, aggregation and attachment to monolayers of chick embryo fibroblast (CEF) cells have been studied using a constant amount of influenza A/fowl plague virus/Rostock/34 (H7N1) and varying amounts of purified mouse polyclonal IgM directed against the haemagglutinin, the major viral neutralization antigen. There are two major types of interaction, (i) At low concentrations of IgM there is aggregation of virus, but no neutralization provided that the aggregates are dispersed by vortexing and dilution. Maximum aggregation occurs at less than seven molecules of IgM per virion and the IgM is probably bound in the ‘staple’ or ‘crab’ conformation at these concentrations, (ii) At higher concentrations there is neutralization and this coincides with inhibition of attachment of virus to CEF cells. Neutralization of 50% infectivity requires about 35 molecules of IgM per virion. The maximum neutralization observed was only 87%. Quantitative data and electron microscopy observations suggest that molecules of IgM at the higher concentrations adopt a planar stance approximately perpendicular to the viral surface. It appears that IgM neutralizes fowl plague virus in vitro primarily by interfering with its attachment to cells; the fraction of neutralized virus that does attach is known not to be internalized.
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Delineation of canine parvovirus T cell epitopes with peripheral blood mononuclear cells and T cell clones from immunized dogs
Three synthetic peptides derived from the amino acid sequence of VP2 of canine parvovirus (CPV) which were recently shown to represent three distinct T cell epitopes for BALB/c mice could prime BALB/c mice for a CPV-specific proliferative T cell response upon immunization. Proliferative responses of peripheral blood mononuclear cells (PBMC) from CPV-immunized dogs upon stimulation with these and other peptides, covering the major part of the sequence of VP2′, identified the presence of T cell epitopes for this species. Most of these epitopes were recognized by PBMC from only a minority of the dogs tested. With three newly generated canine Thyl+ T cell clones, which recognized CPV antigen in association with major histocompatibility complex class II molecules, two distinct T cell epitopes were identified within the unique sequence of VP1.
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Variable regions on the genome of Malawi isolates of African swine fever virus
More LessRestriction enzyme site mapping showed that most BamHI and all ClaI sites were conserved on the genomes of 17 African swine fever virus isolates from separate disease outbreaks that occurred between 1982 and 1989 in Malawi. However, frequent variation between virus genomes did occur due to addition or deletion of DNA sequences at various positions along the genome and 11 virus genotypes could thus be distinguished among the 17 isolates analysed. Length variations occurred at 10 different loci on the virus genome. These variable regions were located between the left DNA terminus and a position up to 48 kb from that terminus, in the centre of the genome 90 to 93 kb from the left DNA terminus and between the right DNA terminus and a position 22 kb from that terminus. Length variations in most of these regions were small (< 1 kb) but variations of about 4 kb occurred in a region up to 20 kb from the left DNA terminus.
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Structural and functional analysis of orthopoxvirus epitopes with neutralizing monoclonal antibodies
More LessNeutralizing monoclonal antibodies (MAbs) were produced in BALB/c mice immunized with live modified vaccinia virus Ankara or infected with sublethal doses of the neurovirulent vaccinia virus strain Munich 1. The immunization scheme proved to be important for obtaining MAbs of different specificity. The MAbs could be classified into three epitope groups (1 A, 1 B and 2). Immunogold electron microscopy demonstrated that the epitopes were localized on the virus surface. In immunoblotting, MAbs were reactive with polypeptides of 14K, 16K and 30K. Purified MAbs binding to the epitopes 1 A and 2 showed a 50 % reduction of 100 p.f.u./0·05 ml vaccinia virus M1 with respectively 3·9 and 5·9 ng of immunoglobulin/ 0·05 ml. MAbs binding to the epitope 1 B neutralized the virus at a concentration of 250 ng/0·05 ml. In intraperitoneal challenge experiments, MAbs binding to the epitopes 1 A and 2 protected mice against 4 LD50 of vaccinia virus Ml, but not against local lesions by subcutaneous application. MAbs against epitope 1 B had no protective effect in vivo. The three epitopes were present in 14 of 16 orthopoxviruses tested but with quantitative differences. Maximal binding (Vmax ) and the antibody concentration at half-maximal binding (Km ) which were calculated as for Michaelis-Menten kinetics from regression analysis of the ELISA data and the MAb concentration giving 50 % plaque reduction were the basis for the evaluation. In monkeypox virus Kopenhagen the epitopes 1 A and 1 B were absent. MAbs binding to epitope 2 reacted just as well as with vaccinia viruses. Ectromelia virus lacked all the epitopes.
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Biochemical and immunological studies of proteins from polydnavirus Chelonus sp. near curvimaculatus
More LessPolydnavirus from Chelonus sp. curvimaculatus (CcV) was purified and 18 structural polypeptides associated with CcV were identified by silver staining. Antibodies were raised against CcV protein and used in testing for the presence of the virus in different tissues of the wasp ovary and in stung eggs. It was also established that the virus does not enter during the first 5 s of oviposition. Furthermore, no degradation of the virus proteins was detected inside the egg within 2 h after oviposition. The glycoprotein nature of virus proteins was also determined by concanavalin A/horseradish peroxidase staining. The amino acid compositions of the most highly abundant peptides (41K, 33K, 21K, 17K and 13K) were determined, as was the N-terminal amino acid sequence of the 41K protein. The latter did not show similarity with any reported protein sequences.
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Evolutionary relationships of virion glycoprotein genes in the S regions of alphaherpesvirus genomes
More LessThe short region in the genome of herpes simplex virus type 1 contains a contiguous array of five genes (US4, US5, US6, US7 and US8) which encode known or proposed virion-surface glycoprotein species. Counterparts for certain of these have been described in the genomes of other alphaherpesviruses, namely herpes simplex virus type 2, pseudorabies virus and varicella- zoster virus. Within each of the US4-, US6- and US7- related sets, the amino acid sequences are most conserved in a region containing several cysteine residues. Comparisons in this region among the three sets were carried out by first aligning three cysteine residues which were very similarly placed in each set, and a number of other similarities were then visible. It was concluded that the US4, US6 and US7 sets of genes are related, and thus have evolved by duplication and divergence. The US8-related sequences are distinct from the US4, US6 and US7 sequences, although possible signs of a distant relationship were detected. The US8 set contains two clusters of cysteine residues, and the sequences around these show some similarity, which was interpreted as evidence for occurrence of an intramolecular duplication event.
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A single amino acid substitution in the large subunit of herpes simplex virus type 1 ribonucleotide reductase which prevents subunit association
More LessThe herpes simplex virus type 1 temperature-sensitive (ts) mutant ts1207 does not induce detectable levels of ribonucleotide reductase activity at the non-permissive temperature (NPT, 39.5 °C). The ts lesion prevents the association of the enzyme's large (RR1) and small (RR2) subunits to give an active holoenzyme and maps within the gene specifying RR1. Here, it is shown that the ts mutant phenotype is due to the substitution of an asparagine for the wild-type (wt) serine at RR1 position 961, which is located within a region highly conserved between herpesviral and cellular RR1 subunit polypeptides. This ts1207 asparagine is predicted to alter a wt α-helix to a β-strand. We have used synthetic oligopeptides, corresponding to the wt amino acid sequence of the mutation site, and antisera raised against them to determine whether this region is involved in subunit association. Neither the oligopeptides nor the antisera inhibit the enzyme activity, or the reconstituted activity formed by mixing intact RR2 and RR1 subunits present in partially purified extracts of cells infected at the NPT with ts1207 or ts1222 (an HSV-1 mutant with a lesion in the RR2 subunit), respectively. We infer from these results that the site of the mutation is unlikely to be positioned at the surface of RR1 and hence is probably not directly involved in subunit association. We suggest that the mutation site identifies an important RR1 region whose alteration in ts1207 changes the structure of a contact region(s) positioned at the RR1/RR2 interface.
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Herpes simplex virus type 1 UL28 gene product is important for the formation of mature capsids
More LessThe herpes simplex virus type 1 temperature-sensitive (ts) DNA-positive mutant ts1203 has been characterized. The ts lesion in ts 1203 was located by marker rescue within the coding region of gene UL28. Nuclei of cells infected with ts1203 at the non-permissive temperature (NPT) contained large numbers of capsids with a uniform morphology. These capsids lacked DNA but had a defined internal structure. No full capsids were detected at the NPT, suggesting that ts1203 was unable to package viral DNA. In this respect ts1203 is similar to ts1201 which has a defect in gene UL26. The capsids made by ts1203 at the NPT, however, contained a more compact internal structure than those of ts1201. In addition, ts1203 capsids were dispersed throughout the nucleus whereas ts1201 capsids were frequently found clustered together in large arrays. Southern blot and sedimentation analyses of viral DNA confirmed that ts1203 had an encapsida- tion defect and showed that most of the mutant DNA at the NPT was of a high Mr. The effect of the ts1203 mutation could not be reversed in the absence of de novo protein synthesis by transferring mutant-infected cells from the NPT to the permissive temperature.
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Possible latent infection with herpes simplex virus in the mouse eye
More LessHerpes simplex virus (HSV) was isolated from organ cultures of anterior segments of the eyes of mice inoculated with virus on the snout or directly onto the cornea at least 5 weeks previously. The frequency of isolation of the virus was not decreased by treatment of the animals with acyclovir, suggesting that the virus is latent by the criteria usually applied. Peroxidase-antiperoxidase staining of organ cultures that had shed virus showed that viral antigens were predominantly present in the anterior uvea. Inoculation of mouse eye anterior segments in vitro showed that this tissue was the most susceptible to productive infection. These results suggest the possibility that HSV can establish a latent infection in tissues of the anterior segment of the mouse eye.
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Murine cytotoxic T lymphocytes specific for herpes simplex virus type 1 recognize the immediate early protein ICP4 but not ICP0
Vaccinia virus recombinants expressing the herpes simplex virus type 1 (HSV-1) genes encoding ICP0 or ICP4 were used to identify the precise target antigen(s) of murine anti-viral cytotoxic T lymphocytes (CTL) specific for the non-structural immediate early proteins. These studies revealed that HSV-1-specific CTL, restricted to class I major histocompatibility complex genes of the H-2k haplotype but not the H-2d or H-2bhaplotypes, would lyse autologous cells expressing ICP4. HSV-1-specific CTL derived from various mice strains failed to lyse target cells expressing ICP0.Calculation of the frequencies of H-2k-restricted virus-specific CTL demonstrated that approximately a third of the total HSV-1-specific response was directed against ICP4. Immunization of mice with either recombinant vaccinia virus or transfected L cells expressing ICP4 induced HSV-1-specific lymphoproli-feration and delayed hypersensitivity but CTLs were not induced. More importantly, such immunized animals were unable to resist or control a subsequent challenge with virulent HSV-1.
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The role of mucosal antibody in immunity to infectious laryngotracheitis virus in chickens
More LessThe role of mucosal antibody in recovery from a primary infection and resistance to reinfection with infectious laryngotracheitis (ILT) herpesvirus was studied in bursectomized chickens, which were unable to synthesize specific antibodies. Viral antigen in the infected trachea was assessed by indirect immunofluorescence on tissue sections and by ELISA. The ability of bursectomized chickens to resolve primary infections as effectively as intact chickens and of vaccinated-bursectomized chickens to prevent the replication of challenge virus without the participation of mucosal antibody, is evidence for the importance of local cell-mediated rather than humoral immune mechanisms in the outcome of infection with ILT virus.
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Characterization of the high M r glycoprotein (gP300) of equine herpesvirus type 1 as a novel glycoprotein with extensive O-linked carbohydrate
The high Mr glycoprotein (gp300) of equine herpesvirus type 1 was found to have an Mr , estimated by SDS-PAGE, of over 400000 and was confirmed as being a surface glycoprotein by 125I-labelling. In contrast to [3H]glucosamine, gp300 showed very low levels of [3H]mannose incoporation. The Mr of gp300 showed no detectable change upon treatment of purified virus with (N-glycanase, and showed only a small change in virus-infected cells treated with tunicamycin. In addition, gp300 failed to bind the lectin concanavalin A. Taken together, these results indicate a lack of N-linked carbohydrate on gp300. The major carbohydrate species were found to be composed primarily of O-linked chains, as indicated by the sensitivity of the protein to monensin, to exoglycanase enzymes specific for sugars present in O-linked chains and to mild alkaline borohydride treatment, which revealed three species of carbohydrate of Mr of > 10000, 2400 and 1100, respectively. Neuraminidase treatment and binding of Helix pomatia lectin indicated the presence of α- N-acetylglucosamine and sialic acid as terminal sugars. Immunological cross-reactivity of gp300 with a high Mr protein of equine herpesvirus type 4 was shown and it also exhibited a marked Mr variation in the vaccine strain Rhinomune.
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The location and nucleotide sequence of the thymidine kinase gene of bovine herpesvirus type 1.2
More LessOn the basis of their restriction endonuclease digestion patterns, four Australian bovine herpesvirus type 1 (BHV-1) isolates were classified as belonging to the BHV-1.2a subtypes. The thymidine kinase (TK) genes of all four BHV-1.2a isolates were located on a 3·5 kb SalI restriction fragment. This is in contrast to North American and European BHV-1.1 isolates whose TK genes are contained on a 2·6 to 2·8 kb salI fragment. The restriction fragments containing the TK genes were cloned into phagemid vectors and their sequences determined using the dideoxynucleotide chain termination method. The BHV-1.2a isolates possessed identical TK gene sequences, which differed from previously published TK sequences for the LA and 6660 BHV-1.1 strains. In addition to five single base alterations, there were six separate base insertions which resulted in two major frameshifts which spanned an area of 72 amino acids or 20% of the expressed TK gene product. The predicted amino acid sequence exhibited a higher degree of similarity to other herpesvirus TKs, suggesting that previously published TK gene sequences may have been incorrect. The present nucleotide sequence and corresponding amino acid composition reinforces previous observations concerning regions of herpesvirus TK amino acid conservation and should assist in future studies into the evolution and functional domains of herpesvirus TKs.
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Construction of a defective adenovirus vector expressing the pseudorabies virus glycoprotein gp50 and its use as a live vaccine
More LessThe gene encoding the pseudorabies virus glycoprotein gp50 was cloned at the very left end of the genome of adenovirus type 5 to give a recombinant adenovirus (Ad-gp50) defective for the E1A gene. Ad-gp50 expressed high levels of gp50 in cells which either complemented (293 cells) or did not complement (Vero and HeLa cells) the E1A gene. Surprisingly, over an extended period, higher levels of gp50 were produced in HeLa cells which lack the E1A gene. Rabbits and mice inoculated with Ad-gp50 showed a strong antibody response against gp50. Some of them were protected from a virulent challenge with pseudorabies virus.
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The structure of the pseudorabies virus genome at the end of the inverted repeat sequences proximal to the junction with the short unique region
More LessThe complete nucleotide sequence is presented of the 2·67 kbp BamHI-EcoRV portion of the the Bam HI 10 fragment of the pseudorabies virus (PRV) genome (strain Ka) containing sequences upstream of the previously reported protein kinase gene, and completing the sequence of this 4008 bp fragment. It is predicted to contain a gene designated RSp40, homologous to gene US1 of herpes simplex virus type 1 (HSV-1), with the potential to encode a protein of 364 amino acids. Analysis of PRV mRNA synthesized in the presence and absence of cycloheximide indicated that, in contrast to its HSV-1 homologue, the PRV gene RSp40 does not specify an immediate-early mRNA. Between the RSp40 gene and the protein kinase gene are two reiterated sequences: one containing 11 tandem copies of a 35 nucleotide sequence and the other containing nine tandem copies of a 10 nucleotide sequence. The BamHI 10 and the BamHI 12 fragments of PRV contain the junctions between the short unique (Us) and short repeat (Rs) regions of the PRV genome. The nucleotide sequence of that portion of the BamHI 12 fragment containing Us sequences was determined so that, by comparison with the nucleotide sequence of the BamHI 10 fragment, the junction between the Us and Rs regions could be defined. In BamHI 10 this was found to be at a point between the two reiterated sequences (which are in the Rs region) and the protein kinase gene (which is in the Us region). The organization of this region of the PRV genome is compared to that of other alphaherpesviruses.
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The gp116 of the gp58/116 complex of human cytomegalovirus represents the amino-terminal part of the precursor molecule and contains a neutralizing epitope
More LessThe glycoprotein complex gp58/116 of human cytomegalovirus (HCMV) represents a dominant antigen for the humoral immune response. We have used the human monoclonal antibody C23, which is capable of neutralizing HCMV in tissue culture without the addition of complement, to study the origin of gp116 as well as the amino acid sequence recognized by the antibody. Our results show that gp116 is derived from the same open reading frame as gp58 and that it represents the amino-terminal portion of the precursor protein. Using prokaryote-expressed β-galactosidase-gp116 fusion proteins, the binding site of C23 was located to between amino acids 27 to 84 of the amino-terminal portion of gp116. Analyses of HCMV-positive human sera revealed that this portion of the molecule is immunogenic during natural infection.
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Nucleotide sequence of a cytomegalovirus single-stranded DNA-binding protein gene: comparison with alpha- and gammaherpesvirus counterparts reveals conserved segments
More LessThe genomic sequence encoding a cytomegalovirus strain Colburn homologue (DB129) of the herpes simplex virus major DNA-binding protein (ICP8) was determined. Multiple alignments of the deduced DB129 amino acid sequence and three alpha- and gammaherpesvirus homologues revealed that 56% of the amino acid residues identical in all four homologues are contained within 12 relatively conserved segments, which together constitute only 11 · 2% of the shortest aligned sequence. In light of published ICP8 deletion analyses, this alignment suggests conserved segments that may participate in forming DNA contacts. The identified conserved regions present interesting targets for site-directed mutagenesis in structure-function analyses.
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Human papillomavirus type 8 contains cis-active positive and negative transcriptional control sequences
More LessHuman papillomavirus type 8 (HPV-8) is one aetiological agent of macular and flat wart-like lesions in patients with epidermodysplasia verruciformis and appears to be closely linked to skin carcinogenesis. A 1.2 kb region of the genome, which was previously shown to contain a viral E2-dependent enhancer, was progressively shortened from both ends with Bal 31. The resulting fragments were tested for their ability to stimulate chloramphenicol acetyltransferase (CAT) expression from the simian virus 40 (SV40) promoter. This analysis showed a complex interaction between cis-active, positive and negative control elements located throughout the non-coding region and the flanking reading frames. Two separate positively acting sequences significantly stimulated expression only in cooperation with a third region, which led to 12-fold, E2-dependent enhancement on its own. A major negative element was not only active in the context of HPV-8 sequences, but also down-regulated SV40 enhancer-promoter-driven CAT expression when cloned downstream of the transcription unit. It acted at the transcriptional level as shown by RNase protection assays and can therefore be regarded as a cis-acting silencer of transcription.
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Restriction maps and sequence homologies of two densovirus genomes
The genomes of Junonia coenia densonucleosis virus (JcDNV) and Galleria mellonella densonucleosis virus (GmDNV) were analysed by restriction endonuclease analysis and Southern blot hybridization. A total of 37 and 33 restriction sites were mapped on JcDNV and GmDNV DNA, respectively. BglI, HaeII and BstEII were site-specific for JcDNV DNA, and BglII and ClaI for GmDNV DNA. The two genomes had nearly identical maps for several restriction endonucleases and Southern blot hybridization using a total genomic JcDNVprobe indicated extensive DNA sequence homologies spanning the entire length of the two genomes. Symmetrical cleavage sites, mapping at the extremities of both genomes, confirmed the presence of inverted terminal repeats of at least 420 to 440 bases in length.
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A synthetic peptide elicits antibody reactive with the native duck hepatitis B virus pre-S protein
More LessSynthetic peptides P37–49 and P63–79, derived from the pre-S region of duck hepatitis B virus (DHBV), have been synthesized. Only P37–49 was reactive with rabbit anti-DHBs/pre-S antibodies by radioimmuno-precipitation. Antiserum prepared against P37–47 reacted with a 35K polypeptide of native DHBs/pre-S by immunoblotting. It is concluded that P37–49 (MGQHPAKSMDVRR) mimics one of the epitopes of the DHBV pre-S antigen.
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Molecular analysis of the haemagglutinin gene of an avian H1N1 influenza virus
More LessThis study presents the first nucleotide sequence and deduced primary amino acid sequence of a subtype H1 haemagglutinin from the avian influenza virus A/duck/Alberta/35/76 (H1N1). The molecule is structurally, antigenically and molecularly similar to H1 haemagglutinins of human viruses but sequence homology differences indicate that there has not been a recent transfer of haemagglutinin genetic information between them.
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Temperature elevation enhances cell surface expression of measles virus fusion protein in infected cells
More LessCell fusion proceeded gradually in measles virus-infected cells incubated at 35 °C. Shift-up of incubation temperature to 39 °C induced rapidly increased cell fusion in spite of the cessation of de novo synthesis of the fusion (F) protein. Pulse-chase experiments showed that there was little difference in the acquisition of immunoreactivity by haemagglutinin (H) and F proteins between the two temperatures. H protein was detected on the cell surface 60 min after the chase at either temperature. However, appearance of F protein on the cell surface took less than 3 h at 39 °C whereas it took 5 h at 35 °C. These data indicate that temperature elevation induces more efficient expression of F protein on the cell surface accompanied by marked syncytium formation in measles virus-infected cells.
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Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector
An improved polyhedrin-based baculovirus expression vector was constructed to expedite distinguishing infections by putative baculovirus recombinants from infections by wild-type (wt) baculovirus. The vector utilizes the Escherichia coli β -galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa califomica nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene were transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (Sf) cells, thereby making the site of the deleted polyhedrin gene available for the insertion and expression of foreign genes under the control of the polyhedrin promoter. Recombinant baculoviruses are readily selected in plaque assays by the development of a blue colour upon the addition of X-Gal. The colour selection renders the retrieval of recombinants less dependent on a high frequency of recombination between the transfer vector and wt baculovirus DNA. The usefulness of this new vector was illustrated by expressing gene I of cauliflower mosaic virus, which encodes a protein of M r 46000. Expression of gene I was at the same level as in cells infected with a conventional polyhedrin-based expression vector. Gene I protein formed large hollow fibre-like structures in the cytoplasm of infected Sf cells. This is the first plant virus protein to be expressed in insect cells by a recombinant baculovirus.
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Genomic characterization of phenotypic variants of beet curly top virus
More LessFull-length infectious DNA clones were constructed for four distinct phenotypic variants of beet curly top virus (BCTV). Southern hybridization assays indicated that each cloned BCTV genome shared sequence homology with pBCT-028, a full-length infectious DNA clone of a California isolate of BCTV previously characterized by others. Restriction endonuclease maps of the cloned BCTV genomes were distinct from one another. Infectivity assays determined that plasmids containing tandem repeats of BCTV genomes were generally more infectious than excised linear DNA inserts. Progeny virus, derived from plants inoculated with cloned DNAs, differed in their ability to infect sugarbeet, Beta vulgaris L., and the severity of symptoms produced in B. vulgaris and other experimental hosts.
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Nucleotide sequence of rice dwarf virus genome segment 4
More LessThe complete nucleotide sequence of rice dwarf virus (RDV) genome segment 4 was determined. Genome segment 4 was 2468 nucleotides long and had a long open reading frame initiating at nucleotides 64 to 66 and terminating at 2245 to 2247. The deduced polypeptide contained 727 amino acid residues with an M r of 79·8K. Amino acid sequences similar to a ‘zinc- finger’ and purine NTP-binding motifs were present in the deduced polypeptide. Considerable amino acid sequence homology was detected between genome segment 4 of RDV and wound tumor virus (WTV). One of the sequences similar to the ‘zinc-finger’ motif was present in a conserved region of the polypeptide of both viruses. However, the sequence similar to the purine NTP-binding motif was not present in the polypeptide encoded by genome segment 4 of WTV.
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A classification of the tobamoviruses based on comparisons among their 126K proteins
More LessThe products of partial proteolysis of the M r 126000 in vitro translation products of the RNA of eight tobamoviruses were separated by SDS-polyacrylamide gel electrophoresis. The peptide patterns obtained were compared using a computer program designed to establish phylogenetic relationships. The resulting most-parsimonious phylogenetic trees grouped the tobamoviruses into clusters I (tobacco mosaic virus, tomato mosaic virus, tobacco mild green mosaic virus, pepper mild mottle virus) and II (sunn-hemp mosaic virus, cucumber green mottle mosaic virus, kyuri green mottle mosaic virus), with ribgrass mosaic virus in an intermediate position. This clustering resembles that obtained when the coat proteins of these viruses are compared. If the tobamoviruses have arisen by divergence from an ancestral type, the results suggest that different parts of the genome have diverged similarly and that recombination has not played a major role in the evolution of the group.
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Antigenic analysis of the coat protein of beet necrotic yellow vein virus by means of monoclonal antibodies
By means of monoclonal antibodies (MAbs), five (groups of) epitopes were identified on particles of beet necrotic yellow vein virus (BNYVV). Epitopes 1 and 2, which were located on the opposite extremities of virus particles, are discontinuous (SDS-labile) epitopes which were destroyed when the particles were treated with trypsin. Epitope 3 is a continuous (SDS-stable) epitope lcoated at the same extremity as epitope 2. It was not destroyed when the particles were treated with trypsin and was present on an Escherichia coli- expressed fusion protein containing amino acids (aa) 1 to 103 of the BNYVV coat protein. The continuous epitope 4, which was located along the entire length of the particles, was found to be present on a fusion protein containing aa 104 to 188 of the BNYVV coat protein but not on trypsin-treated virus particles. In Western blots, these treated particles yielded two slightly smaller coat proteins which failed to react with MAbs specific for epitope 4 but did react with polyclonal antisera and MAbs specific for epitope 3. BNYVV coat protein has a trypsin cleavage site on the carboxyl side of arginine in position 182, so it is therefore suggested that epitope 4 is located on the exposed C terminus, which is composed of aa 183 to 188. Epitope 5 was also located along the entire length of the particles but in a more uneven distribution than epitope 4. This may be because it is a discontinuous epitope that is very sensitive to subtle changes in protein conformation.
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Translation of cucumber necrosis virus RNA in vitro
More LessThe in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analysed in both rabbit reticulocyte lysate and wheatgerm extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of approximate M r 34·6K was produced. In wheatgerm extracts, four proteins of approximate Mr values 41·6K, 34·6K, 24K and 20K were produced. The genomic locations of the CNV in vitro translation products were determined using several experimental approaches including, first, hybrid-arrested translation using negative-sense RNA corresponding to selected regions of the CNV genome, second, in vitro translation of synthetic positive-sense CNV transcripts and third, in vitro translation of CNV virion RNA fractionated according to size. Together these experiments demonstrated that the protein of Mr 34·6K is derived from the 5′-proximal coding region, the 41·6K protein is derived from an internal coding region, and that at least one but probably both the 24K and 20K proteins are derived from the 3′-terminal coding region. In addition, immunoprecipitation of in vitro translation products using anti-CNV polyclonal serum demonstrated that the 41·6K protein is the coat protein. The templates for the expression of CNV cistrons were investigated by in vitro translation of sucrose gradient-fractionated CNV virion RNA as well as in vitro translation of positive-sense synthetic transcripts.
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Nucleotide sequence and evolutionary relationships of cucumber mosaic virus (CMV) strains: CMV RNA 3
More LessThe nucleotide sequence of RNA 3 of two subgroup I strains of cucumber mosaic virus (CMV), Fny-CMV and M-CMV, was determined and compared at both the nucleic acid and protein level with the previously determined, corresponding (partial) sequences of RNA 3 of five other subgroup I strains: C-CMV, D-CMV, I17F-CMV, O-CMV and Y-CMV. Fny-CMV RNA 3 is composed of 2216 nucleotides (nt) and M-CMV RNA 3 2214 nt. Both RNAs contain two open reading frames, the 3a gene and the coat protein gene. These RNAs showed very little nucleotide sequence divergence, either from each other or from the five other subgroup I strains. The nucleotide sequence variation observed was two to 13 differences in the 120 to 123 nt 5′ non-translated regions, six to 17 differences in the 840 nt 3a genes, two to 15 differences in the 296 to 299 nt intergenic regions, three to 25 differences in the 657 nt coat protein genes and two to 10 differences in the 299 to 303 nt 3′ non-translated regions. Protein sequence similarity was also high, with one to four differences in the 279 amino acids of the 3a proteins and two to 13 differences in the 218 amino acids of the coat proteins. Limited nucleotide sequence variation among nine strains of CMV was also shown using an RNA protection assay and a probe specific for Fny- CMV RNA 3. The limited variation shown by RNA 3 of strains of CMV with different passage histories, isolated in different countries over a 50 year period, suggests that the maintenance of the highly conserved nucleotide sequence may be important for other viral RNA functions or interactions.
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Expression of the genome of potato leafroll virus: readthrough of the coat protein termination codon in vivo
I. Bahner, J. Lamb, M. A. Mayo and R. T. HayAn antiserum was raised against a fusion protein containing part of the 56K polypeptide (P5) encoded by the open reading frame (ORF) at the 3′ end of the genome of potato leafroll virus (PLRV). This antiserum reacted specifically with 80K and 90K polypeptides in PLRV-infected protoplasts, with a 90K polypeptide in infected potato tissue and with a 53K polypeptide in protein extracted from purified particles of PLRV. Monoclonal antibodies raised against purified PLRV particles also reacted with these polypeptides, as well as with the 23K coat protein. Virus particles partially purified from infected protoplasts contained some 90K polypeptide as well as the major 23K coat protein. The ORFs of the 23K coat protein and P5 are contiguous and in frame. The results suggest that the P5 polypeptide of PLRV occurs in infected cells as part of a readthrough protein comprising the 23K coat protein joined to the P5 amino acid sequence. Moreover the readthrough protein can be assembled into virus particles as a minor component together with the main 23K component. The P5 protein may thus contribute to properties of PLRV determined by its virus particle surface.
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Ribozymes that cleave potato leafroll virus RNA within the coat protein and polymerase genes
J. W. Lamb and R. T. HayTwo ribozymes were synthesized which were designed to cleave potato leafroll virus (PLRV) positive strand RNA within the regions known to encode the viral coat protein and the predicted RNA polymerase gene. DNA sequences encoding the ribozymes were inserted into the Escherichia coli plasmids pTz 18R and pTz 19R under the control of the bacteriophage T7 promoter and enzymically active RNA molecules generated by transcription by T7 RNA polymerase in vitro. Each ribozyme cleaved its cognate site in RNA derived from either cDNA or PLRV particles. Ribozyme cleavage sites within the polymerase gene and coat protein gene were determined and shown to be at the predicted sequence immediately downstream from a GUC motif. An altered version of the ribozyme which recognized the sequence in the coat protein gene was isolated in which a single adenosine residue in the enzymic loop of the ribozyme was deleted. This mutated ribozyme was unable to cleave RNA molecules containing the coat protein ribozyme target site.
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Characterization of a potato leafroll luteovirus subgenomic RNA: differential expression by internal translation initiation and UAG suppression
More LessNorthern blot analysis of Solanum tuberosum infected with potato leafroll luteovirus revealed the 6 kb genomic RNA and a major 2.3 kb subgenomic RNA. The 5′ end of the subgenomic RNA was located at nucleotide 3653 in an intergenic region located at the centre of the viral genome upstream of three open reading frames (ORFs). Transient expression in tobacco and potato protoplasts of the β-glucuronidase reporter gene fused to various putative regulatory sequences present in the subgenomic RNA was used to study their influence on expression levels. We observed a suppression of the amber stop codon separating the coat protein (CP) gene from a downstream ORF (56K protein), to a level of 0.9% to 1.3%. Translation initiation at the AUG of an ORF (17K protein) which is nested within the CP gene, exceeds translation of the CP gene itself by a factor of 7.
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Potato virus Y helper component protein is associated with amorphous inclusions
D. A. Baunoch, P. Das and V. HariThe distribution of the helper component (HC) protein of potato virus Y in tissues and cells of infected plants was studied by immunoblotting and immunogold labelling techniques. This HC protein was found in leaf blade and vein tissue but not in the petiole of leaves. In infected cells, the protein was localized in rod-shaped cytoplasmic inclusions known as amorphous inclusions.
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